The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class bound to an uncommon 18 base-pairDNA consensus segment have been studied. In addition, the dynamic behavior of the wild type and selected mutants have been characterized. Structural changes in the homeodomain upon mutation are accompanied by corresponding changes in the motional properties of the molecule. One also observes low frequency motions in the wild type analog that are indicative of thermal unfolding. These results imply that the initiation of thermal unfolding is accompanied by low frequency exchange between the folded and unfolded states. Thermodynamic measurements on the same wild type and mutant homeodomains has been completed. The change in the heat capacity with temperature, as well as the enthalpies and entropies of DNA binding were analyzed in detail. The results imply that the initiation of the thermal unfolding process involves low frequency exchange motion.A detailed investigation of a human member of the NK-2 class of homeodomains, NKX-2.5 has been initiated. Studies include a determination of the three-dimensional structure of the full length protein (331 residues) as well as the wild type and mutant analogs of the homeodomain. Mutations within the homeodomain are directly associated with several forms of congenital heart disease. We have shown that these mutations associated with disease all result in either significant structural alterations of the homeodomain or in its DNA binding behavior.